In vitro culture techniques have limitations that must be overcome to increase the feasibility of cell-based tissue engineering strategies. A limitation in static culture is insufficient transport of oxygen and other nutrients to regions more than a few hundred microns from the scaffold surface, which leads to nonhomogenous cell distribution and extracellular matrix production. See Ishaug, S. L. et al. (1997) “Bone formation by three dimensional stromal osteoblast culture in biodegradable polymer scaffolds,” J Biomed Mater Res 36:17; Yu et al. (2004) “Bioreactor-based bone tissue engineering: the influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization,” Proc Natl Acad Sci USA 101: 11203; Gomes et al. (2003) “Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starchbased three-dimensional scaffolds,” J Biomed Mater Res Part A 67A: 87; Volkmer et al. (2008) “Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone,” Tissue Eng Part A 14:1331; and Martin et al. (2004) “The role of bioreactors in tissue engineering,” Trends Biotechnol 22:80. Conventional bioreactor systems attempt to overcome these limitations by increasing nutrient transfer to cells via dynamic culture. Further, mechanical stimulation through fluid shear stresses has been shown to be influential on bone differentiation and mineralization. See Bilodeau et al. (2006) “Bioreactors for tissue engineering: focus on mechanical constraints. A comparative review,” Tissue Eng 12:2367; Bancroft et al. (2003) “Design of a flow perfusion bioreactor system for bone tissue-engineering applications,” Tissue Eng 9:549; Bancroft et al. (2002) “Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner,” Proc Natl Acad Sci USA 99:12600.
Another significant challenge in the implementation of cell based tissue engineering strategies remains the inability to successfully culture large constructs in vitro. One approach to overcoming this difficulty is a bottom up approach to creating a tissue engineering construct. A unitary polymer scaffold is constructed in its final shape and seeded with cells. The cells are then cultured in vitro to allow for proliferation and matrix deposition throughout the scaffold. However, such bottom up approaches are limited by the scaffold size and cell density that will allow for homogenous growth and matrix production throughout the scaffold. For example, central oxygen concentration of cells cultured in scaffolds 9 mm by 5 mm were shown to drop to 0% after just five days of culture. Bioreactor culture mitigate this effect. However, central oxygen concentration of the same constructs cultured in a conventional perfusion bioreactor were only 4%.
Some previous studies focus on bioreactor systems as a means to culture cells for bone tissue engineering purposes. See Meinel et al. (2004) “Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow,” Ann Biomed Eng 32:112; Sikavitsas et al. (2003) “Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces,” Proc Natl Acad Sci USA 100:14683; Sikavitsas et al. (2005) “Flow perfusion enhances the calcified matrix deposition of marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds,” Ann Biomed Eng 33:63; Sikavitsas et al. (2002) “Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor,” J Biomed Mater Res 62:136; van den Dolder et al. (2003) “Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh,” J Biomed Mater Res Part A 64A:235; Grayson et al. (2008) “Effects of initial seeding density and fluid perfusion rate on formation of tissue-engineered bone,” Tissue Eng Part A 14:1809; Janssen et al. (2006) “A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: in vivo bone formation showing proof of concept,” Biomaterials 27:315.
Several different types of bioreactor systems have been investigated, including spinner flasks (Stiehler et al. (2009) “Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells,” J Biomed Mater Res Part A 89A:96), rotating wall bioreactors (Wang et al. (2009) “Regulation of adult human mesenchymal stem cells into osteogenic and chondrogenic lineages by different bioreactor systems,” J Biomed Mater Res Part A 88A:935), and perfusion systems (Gomes et al. (2006) “Bone tissue engineering constructs based on starch scaffolds and bone marrow cells cultured in a flow perfusion bioreactor,” Adv Mater Forum III 514:980; Holtorf et al. (2005) “Flow perfusion culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexamethasone,” J Biomed Mater Res Part A 72A:326; Datta et al. (2006) “In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation,” Proc Natl Acad Sci USA 103:2488). Spinner flask and rotating wall bioreactor systems are effective at creating a homogenous media solution on the exterior of the scaffold, but do not effectively perfuse media into the scaffold.
Perfusion systems have been demonstrated to perfuse media throughout the scaffold and have been shown to upregulate osteoblastic markers and increase calcium deposition. In a study utilizing a perfusion bioreactor, flow rate was shown to increase both the calcium matrix deposition and the rate of late osteoblastic differentiation as shown by osteopontin (OPN) expression. See Bancroft et al. (2002), supra, Proc Natl Acad Sci USA 99:12600. Although conventional perfusion systems typically enhance the flow of media to the center of the scaffold, they require custom-made parts and specific scaffold design to successfully perfuse media into the scaffold, making them difficult to fabricate and use.